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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Assessment of Nerve Stimulation-Induced Release of Purines from Mouse Kidneys by Tandem Mass Spectrometry

Center for Clinical Pharmacology, Departments of Medicine (J.R., Z.M., E.K.J.) and Pharmacology (E.K.J.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Received February 6, 2008; accepted March 3, 2008.

	Abstract

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Adenosine, formed from AMP and metabolized to inosine, modulates renal sympathetic neurotransmission. The present study had two goals: 1) to develop ultrasensitive and specific mass spectrometry-based assays for cAMP, AMP, adenosine, inosine, and, for comparison, guanosine using state-of-the-art tandem liquid chromatography-mass spectrometry (LC-MS-MS); and 2) to quantify the effects of renal sympathetic nerve stimulation on the release of cAMP, AMP, adenosine, inosine, and guanosine from the isolated, perfused mouse kidney. Using LC-MS-MS, we developed highly sensitive (detection limit of 0.02–0.05 pg/µl) and accurate (r² > 0.99) assays for all the aforementioned compounds. In the perfused mouse kidney (n = 9), periarterial (renal sympathetic) nerve stimulation elicited a frequency-dependent (0, 3, 5, 7, and 9 Hz) and significant (p = 0.0148, repeated measures analysis of variance) increase in the concentration of inosine in the renal venous perfusate (29 ± 8, 51 ± 8, 54 ± 11, 65 ± 15, and 80 ± 20 pg/µl, respectively), yet concomitantly decreased (p = 0.0239, repeated measures analysis of variance) the concentration of AMP in the renal venous perfusate (3.8 ± 1.3, 3.2 ± 1.7, 2.4 ± 1.5, 2.0 ± 1.1, and 1.1 ± 0.4 pg/µl, respectively). No significant changes were observed in the levels of adenosine, cAMP, or guanosine in the renal venous perfusate. These results indicate that using state-of-the-art mass spectrometric methods, it is possible to investigate trace amounts of purines released from mouse organs in perfusion and that renal sympathetic nerve stimulation is associated with a robust increase in the main metabolite of adenosine (inosine), while concomitantly decreasing AMP. This suggests that renal sympathetic nerve stimulation influences the efficiency of AMP conversion to adenosine and hence to inosine.

Along these lines, adenosine may be an important regulator of the effects of renal sympathetic nerves on kidney function. ATP is a cotransmitter in the sympathetic nervous system and is stored and released from vesicles by exocytosis along with norepinephrine (Burnstock, 1995). ATP can be metabolized to AMP (the immediate precursor to adenosine) in the neuroeffector junction (Westfall et al., 2002); moreover, β-adrenoceptors can participate in the production of adenosine in response to renal sympathetic nerve stimulation (Mi and Jackson, 1999), perhaps by releasing cAMP (an immediate precursor to AMP) (Mi and Jackson, 1995; Jackson and Mi, 2000; Jackson and Dubey, 2001). Adenosine acts on the sympathetic postganglionic prejunctional membrane to inhibit the release of norepinephrine from renal sympathetic nerves (Hedqvist and Fredholm, 1976; Hedqvist et al., 1978; Ekas et al., 1981). Yet, postjunctionally, adenosine enhances renal vascular responses to activation of renal sympathetic nerves (Hedqvist and Fredholm, 1976; Hedqvist et al., 1978) and augments the renal vascular response to angiotensin II (Modlinger and Welch, 2003). Furthermore, adenosine increases sodium chloride reabsorption (Vallon et al., 2006), particularly in the proximal tubule, and this effect may augment antinatriuresis induced by stimulation of renal sympathetic nerves. Finally, adenosine stimulates the sympathetic nervous system by activation of renal afferent nerves (Katholi et al., 1984), a process that may contribute to the pathophysiology of hypertension (Katholi et al., 1985).

Despite the likelihood that adenosine participates in the renal response to sympathetic nerve activity, data addressing the effects of renal sympathetic nerve stimulation on renal purine release are quite limited. In this regard, a study by Fredholm and Hedqvist (1978) demonstrates nerve stimulation-induced release of radiolabeled adenosine from rabbit kidneys preloaded with [³H]adenine, and a report by Mi and Jackson (1999) shows release of adenosine by renal nerve stimulation in the rat kidney.

The role of adenosine in the regulation of renal function by renal nerve stimulation could perhaps best be addressed in mouse kidneys because mice null for all of the subtypes of adenosine receptors and some of the enzymes involved in adenosine biosynthesis are currently available (Yaar et al., 2005). However, it is unknown whether renal nerve stimulation affects purine release from the mouse kidney.

The goal of the present study was 2-fold: 1) to develop ultrasensitive and specific mass spectrometry-based assays for cAMP, AMP, adenosine, inosine, and guanosine using state-of-the-art LC-MS-MS (TSQ Quantum Ultra; Thermo Electron Corporation, Waltham, MA); and 2) to apply our new assay to quantify the effects of renal sympathetic nerve stimulation on the release of cAMP, AMP, adenosine, inosine, and guanosine from the isolated, perfused mouse kidney.

	Materials and Methods

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Animals. Male C57BL mice (n = 9) were obtained from Taconic Farms (Germantown, NY) and used for experiments at approximately 6 to 8 weeks of age. Animals were housed at the University of Pittsburgh Animal Facility and fed Pro Lab RHM 3000 rodent diet (PMI Feeds, Inc., St. Louis, MO). The Institutional Animal Care and Use Committee approved all procedures.

Perfusion of Mouse Kidneys. Mice were anesthetized with Inactin (100 mg/kg i.p.), the right ureter was ligated near the bladder, and the bladder was cannulated with PE-50 tubing to allow urine to drain from the left kidney. The aorta and vena cava were cleared above and below the left renal artery, the distal aorta and vena cava were cannulated with PE-10 and PE-50 tubing, respectively, and these cannulas were advanced as near as possible to the origins of the left renal artery and vein. Tyrode's solution was perfused through the PE-10 tubing during this procedure to maintain renal perfusion during surgical procurement of the left kidney. All vessels branching from the aorta and vena cava near the left renal artery and renal vein were ligated, and the aorta and vena cava just proximal to the left renal artery and vein were ligated.

The left kidney was transferred to a Hugo Sachs Elektronik-Harvard Apparatus GmbH (March-Hugstetten, Germany) kidney perfusion system. This system included the following components: model UP 100 Universal Perfusion System, model ISM 834 Channel Reglo Digital Roller Pump, a glass double-walled perfusate reservoir, an R 120144 glass oxygenator, mechanical integration of the oxygenator with the Universal Perfusion System UP 100, a Windkessel for absorption of pulsations, an inline holder for disc particle filters (80 µm), a temperature-controlled Plexiglas kidney chamber integrated with the UP 100, and a thermostatic circulator. The Plexiglas chamber contained a heat exchanger to maintain the temperature of the perfusate at 37°C at the point of entry into the tissue and also contained a device to extract bubbles from the perfusate just before the perfusate entered the kidney.

The Tyrode's solution (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 1.1 mM MgCl₂, 12 mM NaHCO₃; 0.42 mM NaH₂PO₄, 5.6 mM D(+)-glucose, pH 7.4) was maintained at 37°C in the double-walled perfusate reservoir, bubbled with 95% oxygen/5% carbon dioxide, and pumped by the roller pump through a glass oxygenator (95% oxygen/5% carbon dioxide), through an inline particle filter, through an inline Windkessel, through a heat exchanger, through an inline bubble remover, and finally through the kidney. Kidneys were perfused (single-pass mode) at a constant flow (1.5 ml/min). Perfusion pressure was monitored with a Statham pressure transducer (model P23ID; Statham Division, Gould Inc., Oxnard, CA) and recorded on a Grass model 79D polygraph (Grass Instruments, Quincy, MA).

After a 2-h rest period, renal sympathetic nerve activation was accomplished by placing bipolar electrodes around the renal artery and electrically stimulating the periarterial sympathetic nerves with a Grass stimulator (model SD9E) using bipolar square wave pulses (pulse duration, 1 ms; electrical potential, 35 V). Increasing frequencies of nerve stimulation (0, 3, 5, 7, and 9 Hz) were applied for 5 min, and during the last minute of renal sympathetic nerve stimulation, perfusate exiting the renal vein was collected on ice and immediately frozen at -40°C for later analysis of purines.

Measurement of cAMP, AMP, Adenosine, Inosine, and Guanosine by Mass Spectrometry. AMP, cAMP, adenosine, inosine, and guanosine were purchased from Sigma-Aldrich (St. Louis, MO). The internal standard ([10-¹³C]adenosine) was from Medical Isotopes Inc. (Pelham, NH). All standards were stored at -20°C, and the internal standard was stored at -80°C. Solutions of standards (50 ng/µl) and internal standard (1 ng/µl) were prepared in ultrapure water and stored at -20°C. The mixed solution of purines (1 ng/µl) was prepared each day by dilution in ultrapure water and kept at 4°C. Additional dilutions of standards were prepared from this solution by serial dilution. Methanol (for LC-MS-MS) was from Riedel-Dehae (Seelze, Germany), and analytical grade formic acid was from Fluka (Buchs, Switzerland).

AMP, cAMP, adenosine, inosine, and guanosine were quantified with selective reaction monitoring using the Thermo Electron TSQ Quantum-Ultra system and the HESI source. The high-pressure liquid chromatography (HPLC) column was an Agilent Zorbax eclipse XDB-C-18 column (3.5-µm beads; 2.1 x 100 mm), and the mobile phase consisted of linear gradient changes involving two buffers: buffer A was 0.1% formic acid in water, and buffer B was 0.1% formic acid in methanol. The mobile phase flow rate was 300 µl/min. The gradient (A/B) was as follows: 0 to 2 min, 98.5/1.5%; 2 to 4 min, to 98/2%; 5 to 6 min, to 92/8%; 7 to 8 min, to 85/15%; and 9 to 11.5 min, to 98.5/1.5%. Column temperature was kept at 20°C.

For maximal sensitivities, AMP and adenosine were used for ptimization of parameters of the ion source. In this regard, two separate tune files were used in the process of determination; 0 to 4.5 min, tune file 1 for the monitoring of AMP ions; and 4.5 to 11.5, tune file 2 for monitoring of ions of cAMP, adenosine, internal standard, inosine, and guanosine. The following parameters were the same in the TSQ tune files 1 and 2: ion spray voltage, 3800 kV; ion transfer tube temperature, 270°C; source vaporization temperature, 220°C; Q2 collision-induced dissociation gas, argon at 1.5 mTorr; sheath gas, nitrogen at 50 psi; auxiliary gas, nitrogen at 40 psi; Q1/Q3 width, 0.7/0.7 atomic mass units; source collision-induced dissociation, off; scan width, 0.5 atomic mass units; and scan time, 0.05 s. The tube lens offset was 131 V for tune file 1 and 123 V for tune file 2.

Six selective reaction monitoring transitions were monitored: 348 136 for AMP with a collision energy of 21 V, 268 136 for adenosine with a collision energy of 19 V, 278 141 for [10-¹³C]-adenosine as internal standard with a collision energy of 19 V, 269 137 for inosine with a collision energy of 20 V, 284 152 for guanosine with a collision energy of 21 V, and 330 136 for cAMP with a collision energy of 28 V. Calibration standard curves standards were constructed at concentrations of 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 pg/µl in ultrapure water and Dulbecco's phosphate saline (Invitrogen, Carlsbad, CA). Each concentration was injected four to eight times.

Statistics. Data were analyzed by repeated measures one-factor analysis of variance. The criterion of significance was p < 0.05. All values in text and figures are means ± S.E.M.

	Results

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Figure 1 shows typical chromatograms for cAMP, AMP, adenosine, [10-¹³C]adenosine, inosine, and guanosine. Table 1 shows average retention times for all analytes. All analytes of interest eluted in less than 8 min, thus rendering the assays very rapid. For adenosine, inosine, and guanosine, the detection limit (signal/noise ratio of greater than 3) was 0.02 pg/µl using an injection volume of 20 µl. For AMP and cAMP, the detection limit was slightly higher (0.05 pg/µl). Figure 2 illustrates typical standard curves for AMP and cAMP, and Fig. 3 shows typical standard curves for adenosine, inosine, and guanosine. These curves were generated with eight repeat injections at each level of standard, and the r² values were always greater than 0.99. As shown in Table 2, the coefficients of variation for repeat injections of standards were low (1.1–12.9%), averaging 5.2% for all compounds and concentrations. The assay was unbiased as evidenced by the fact that when nominal concentrations of 5 pg/µl of AMP, cAMP, adenosine, inosine, and guanosine were injected, the values calculated from the standard curves were 5.0 pg/µl.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Typical chromatograms for AMP, cAMP, adenosine, [10-13C]adenosine, inosine, and guanosine. x-Axis is time (minutes), and y-axis is ion current (arbitrary units).

View larger version (6K): [in this window] [in a new window]   Fig. 2. Standard curves for AMP and cAMP. x-Axis is concentration (picograms per microliter), and y-axis is peak area for analyte divided by peak area for internal standard (IS).

View larger version (10K): [in this window] [in a new window]   Fig. 3. Standard curves for adenosine, inosine, and guanosine. x-Axis is concentration (picograms per microliter), and y-axis is peak area for analyte divided by peak area for internal standard (IS).

Basal levels of AMP, cAMP, adenosine, inosine, and guanosine in the venous perfusate from the mouse kidney were 3.8 ± 1.3, 0.33 ± 0.25, 26 ± 5, 29 ± 8, and 4.4 ± 0.7 pg/µl, respectively. Periarterial nerve stimulation significantly (p < 0.0001) increased renal perfusion pressure in a frequency-related fashion (Fig. 4). In contrast, nerve stimulation significantly (p = 0.0239) decreased the renal venous perfusate levels of AMP (the immediate precursor of adenosine; Fig. 5). Although periarterial nerve stimulation did not alter renal venous levels of adenosine (26 ± 5, 29 ± 8, 22 ± 8, 19 ± 8, and 19 ± 9 at 0, 3, 5, 7, and 9 Hz, respectively), periarterial nerve stimulation markedly and significantly (p = 0.0148) increased renal venous perfusate levels of inosine (the immediate metabolite of adenosine; Fig. 6). Renal nerve stimulation did not alter renal venous levels of guanosine (4.4 ± 0.7, 4.7 ± 0.9, 3.6 ± 0.9, 3.4 ± 0.9, and 4.0 ± 1.1 at 0, 3, 5, 7, and 9 Hz, respectively) or cAMP (0.33 ± 0.25, 0.35 ± 0.12, 0.18 ± 0.05, 0.09 ± 0.03, and 0.10 ± 0.04 at 0, 3, 5, 7, and 9 Hz, respectively).

View larger version (10K): [in this window] [in a new window]   Fig. 4. Effects of renal nerve stimulation on perfusion pressure in the perfused mouse kidney. Values represent means ± S.E.M. for n = 9, and p value is from repeated measures analysis of variance. a, p < 0.05 versus 0 Hz (Fisher's LSD test).

View larger version (8K): [in this window] [in a new window]   Fig. 5. Effects of renal nerve stimulation on the release of AMP from the perfused mouse kidney. Values represent means ± S.E.M. for n = 9, and p value is from repeated measures analysis of variance. a, p < 0.05 versus 0 Hz (Fisher's LSD test).

View larger version (8K): [in this window] [in a new window]   Fig. 6. Effects of renal nerve stimulation on the release of inosine from the perfused mouse kidney. Values represent means ± S.E.M. for n = 9, and p value is from repeated measures analysis of variance. a, p < 0.05 versus 0 Hz (Fisher's LSD test).

	Discussion

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In our previously published studies, we report using a variety of analytical approaches to measure adenosine, adenosine precursors, and adenosine metabolites, including microbore HPLC with ultraviolet detection (Jackson and Ohnishi, 1987), conventional HPLC with ultraviolet detection (Mi and Jackson, 1995), HPLC with fluorescence detection (Jackson et al., 1996), and, most recently, HPLC coupled to an ion-trap mass spectrometer (Jackson et al., 2006). Our experience is that all of the aforementioned methods are, in one aspect or another, less than ideal. HPLC with ultraviolet detection is convenient and relatively inexpensive but suffers from limited sensitivity and specificity with multiple interfering peaks except in the cleanest of samples. Coupling HPLC with fluorescence detection greatly reduces background noise and increases sensitivity but is limited to purines that can form the fluorescent etheno derivative (Jamal et al., 1988). HPLC coupled with mass spectrometry out-performs both ultraviolet and fluorescence detection methods because unlike fluorescence detection, a wide range of purine analytes can be targeted in a single run, and unlike ultraviolet detection, sensitivity and specificity are relatively high. In this regard, we report using a Thermo Electron HPLC coupled to a Thermo Electron LCQ Duo ion trap mass spectrometer to measure purines in the rat kidney (Jackson et al., 2006), and this technique is quite adequate for that task. However, quantification of purines in the mouse kidney is a more demanding analytical challenge. Our HPLC-ion trap mass spectrometry method has a sensitivity of approximately 25 pg of adenosine injected on-column (Jackson et al., 2006), which is borderline with regard to the sensitivity required for measuring purines in perfusate from mouse kidneys.

Accordingly, we developed yet another method of measuring purines that uses the extreme sensitivity of the Thermo Electron TSQ Quantum-Ultra mass spectrometer. This method affords a sensitivity of 0.4 pg of adenosine injected on-column (0.02 pg/µl with an injection volume of 20 µl) and therefore is approximately 62.5 times more sensitive than our previously reported method using the LCQ Duo ion trap mass spectrometer. This method also gives rise to highly linear standard curves, low coefficients of variation, and rapid sample processing times.

The main disadvantage of our current method is cost. The instrument is expensive, the maintenance/service contract is costly, and for optimal performance, a highly trained and skilled analytical chemist is required to tune and clean the instrument and conduct and trouble-shoot the assays. In addition, considerable resources are required for internal standards, HPLC columns, high-quality gasses, and disposable vials. Therefore, the assays are best developed and maintained as an institutional resource to be shared by multiple users both within and across institutions.

Some care has to be exercised with regard to the sample matrix. For example, samples cannot be collected in perchloric acid, a popular method for rapid inactivation of enzymes in samples. In our experience, even with careful neutralization, perchloric acid severely suppresses ionization in the ion source such that assay sensitivity is badly compromised. However, collection of samples in organic solvents (for example acetonitrile) to denature and precipitate proteins/enzymes does not interfere with the assay and will facilitate sample concentration if required. Although perchloric acid compromises sensitivity, the assay would be expected to perform adequately with a wide range of biological samples including dialysate, urine, plasma, and cell and tissue extracts.

One goal of the current study was to develop purine assays using LC-MS-MS for measuring purine release from mice organs and tissues, and that goal was accomplished. A second objective was to apply our new method to determine whether renal periarterial (renal sympathetic) nerve stimulation elicits a frequency-dependent change in the release of purines from the isolated, perfused mouse kidney.

The results of this study clearly demonstrate that renal sympathetic nerve stimulation causes a profound increase in release of inosine into the renal venous perfusate. In fact, our results show that renal sympathetic nerve stimulation can increase inosine release into the renal venous perfusate by approximately 3-fold. The lack of change in guanosine levels in the renal venous perfusate provides assurance that the observed increases in inosine levels are not due to nonspecific processes such as organ degradation due to time or nerve stimulation-induced damage. We also observe that the increase in inosine release into the renal venous perfusate is not accompanied by augmentation of adenosine release into the renal venous perfusate. This indicates that the isolated, perfused mouse kidney efficiently (quantitatively) metabolizes adenosine to inosine so that all of the adenosine released in response to sympathetic nerve stimulation is converted to inosine before reaching the renal venous circulation.

The range of frequencies of renal nerve stimulation in the present study corresponds to that observed in vivo from the physiological to the high pathophysiological (DiBona and Kopp, 1997). Each kidney was exposed to all levels of stimulation. However, to avoid desensitization, we were careful to go from low to high frequencies (not the other way around), and we kept the time of stimulation at each frequency to 5 min. In pilot experiments, we repeated the nerve stimulation and observed the same level of vasoconstriction, so desensitization apparently did not occur.

It is interesting to note that higher frequencies of renal nerve stimulation continue to drive inosine levels upward, yet changes in perfusion pressure plateau. It may be that as the frequency of renal nerve stimulation is increased, norepinephrine saturates -adrenoceptors so that vasoconstriction is maximal, yet the prejunctional sympathetic nerve terminals continue to release adenine nucleotides in a frequency-related fashion.

Another important finding of the current study is that renal sympathetic nerve stimulation markedly lowers the concentration of AMP in the renal venous perfusate. In fact, AMP levels in the renal venous perfusate are approximately 3-fold higher under basal, unstimulated conditions compared with AMP levels observed at the highest frequency of renal sympathetic nerve stimulation. This finding can be readily integrated into previously published results showing rapid protein kinase C-dependent activation of ecto-5'-nucleotidase (CD73) by ₁-adrenoceptor activation (Kitakaze et al., 1995). Inasmuch as renal sympathetic nerve stimulation would release endogenous norepinephrine, which would stimulate ₁-adrenoceptors, and ecto-5'-nucleotidase converts AMP to adenosine, it seems quite reasonable to attribute the reduction in AMP and the concomitant increase in inosine (the main adenosine metabolite) in part to renal nerve stimulation-induced activation of ecto-5'-nucleotidase. In addition, activation of sympathetic nerves releases nucleotidases into the neuroeffector junction, and these releasable enzymes may then degrade adenine nucleotides to adenosine (Westfall et al., 2002). The implication therefore is that renal sympathetic nerve stimulation efficiently activates the renal adenosine system, which may have important consequences for renal function.

As discussed, renal nerve simulation decreases AMP levels and increases inosine levels. However, the absolute mass increase in inosine is higher than the absolute mass decrease in AMP. It is possible that the levels of AMP are determined at steady state by the balance between the rate of AMP formation versus the rate of AMP metabolism to adenosine. On the other hand, because inosine is often the ultimate metabolite of purines in perfused tissues, inosine accumulates and does not come to steady state, and so the levels of inosine are affected mainly by the mass flux through the pathway. Although sympathetic nerve stimulation would be expected to release adenine nucleotides, this does not increase AMP levels because AMP metabolism is accelerated but does increase inosine levels because of the increased mass flux of purines through the metabolic pathway.

It is possible that at least some of the inosine exiting the kidney in response to renal nerve stimulation is derived from purines released from sympathetic nerve varicosities because ATP is well known to be coreleased with norepinephrine (Westfall et al., 2002). However, it is certainly possible that some of the purines are coming from postjunctional sites due to the effects of norepinephrine on -adrenoceptors or β-adrenoceptors (Mi and Jackson, 1999). However, to clarify this issue more precisely, additional follow-up experiments with adrenergic blockers to inhibit postjunctional effects are required.

The present study shows that, at least in the mouse kidney, renal sympathetic nerve stimulation does not increase renal venous levels of cAMP. This is not entirely unexpected. Indeed, renal sympathetic nerve stimulation would (via norepinephrine) stimulate renal β-adrenoceptors coupled to adenylyl cyclase, and transport mechanisms would escort intracellular cAMP to the extracellular compartment (Jackson and Raghvendra, 2004). However, because protein kinase C activates both ectophosphodiesterase (Jackson and Mi, 2008) and ecto-5'-nucleotidase (Kitakaze et al., 1995), this extracellular cAMP would be readily converted to AMP, adenosine and hence to inosine. Therefore, inosine seems to be the main downstream metabolite of purine mobilization in the mouse kidney induced by renal sympathetic nerve stimulation.

In summary, the present study validates an ultrasensitive, specific, rapid, accurate, and convenient method to monitor purine release by the isolated, perfused mouse kidney. Indeed, as far as we know, this assay represents the most sensitive assay ever reported for purines. Using this method, we demonstrate the feasibility of investigating purine release from the isolated, perfused mouse kidney in response to renal sympathetic nerve stimulation at physiological frequencies. Our studies show that renal sympathetic nerve stimulation augments the disappearance of AMP and stimulates the appearance of inosine, thus suggesting a nerve stimulation-induced realignment of purine metabolism toward the biologically active nucleosides. To explore the role of adenine nucleotides and nucleosides in cardiovascular and renal physiology and pharmacology, it will be necessary to use genetically altered mice. This entails small animals and small organs and, consequently, small samples with low levels of purines. Our assays will permit investigators to overcome these analytical barriers. Thus, this work provides a foundation for future studies to explore biochemical mechanisms that govern the fate of purines during renal sympathetic nerve stimulation.

	Footnotes

 
This study was supported by the National Institutes of Health Grants HL69846 and DK68575.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.137752.

ABBREVIATIONS: LC-MS-MS, tandem liquid chromatography-mass spectrometry; HPLC, high-pressure liquid chromatography; LSD, least significant difference.

Address correspondence to: Dr. Edwin K. Jackson, Center for Clinical Pharmacology, Departments of Medicine and Pharmacology, University of Pittsburgh School of Medicine, 100 Technology Drive, Suite 450, Pittsburgh, PA 15219-3130. E-mail: edj{at}pitt.edu

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